Biofuels from Algae || Respirometric Balance and Carbon Fixation of Industrially Important Algae

Biofuels from Algae

C H A P T E R

4

Respirometric Balance and CarbonFixation of Industrially Important

AlgaeEduardo Bittencourt Sydney, Alessandra Cristine Novak, Julio

Cesar de Carvalho, Carlos Ricardo SoccolBiotechnology Division, Federal University of Parana, Curitiba, Brazil

4.1 INTRODUCTION

The Framework Convention on Climate Change, signed in Rio de Janeiro in 1992, madeglobal warming a major focus, and the development of technologies for reducing/absorbinggreenhouse gases (GhG) gained importance. After another 20 years, at Rioþ20, the finaldocument stated clear concern about emissions and the need to reduce them by 2020.

Rubin et al. (1992) divided the GhG reduction alternatives into three groups: conservation,direct mitigation, and indirect mitigation. Conservation measures reduce electricity con-sumption and thus GhG emissions; direct mitigation techniques capture and remove CO2

emitted by specific sources; and indirect mitigation involves offsetting actions in whichGhG producers support reductions in GhG emission.

The concept behindmost disposal methods is to offset the immediate effect on the levels ofcarbon dioxide in the atmosphere by relocation, i.e., by injection into either geologic oroceanic sinks (Stewart and Hessami, 2005). Relocation in ocean and deep saline formationshas the capacity for 1012 tons of CO2, whereas global carbon dioxide emissions in 2009 were33�106 tons (Olivier et al., 2011), which means 30,000 years of relocation. Problems related tothis issue are the unknown possible environmental problems (such as acidification, forexample), costs, and the necessity to concentrate CO2 before relocation (how will it workto transport CO2 emissions, for example?).

67 # 2014 Elsevier B.V. All rights reserved.

68 4. RESPIROMETRIC BALANCE AND CARBON FIXATION OF INDUSTRIALLY IMPORTANT ALGAE

Therefore, long-term mitigation technologies for CO2 and other GhG gas removal came tobe developed. They can be generally classified into two categories: (1) chemical reaction-based technologies and (2) biological CO2 mitigation.

Chemical reaction-based CO2 mitigation approaches are energy-consuming and costlyprocesses (Lin et al., 2003), and the only economical incentive for CO2 mitigation using thechemical reaction-based approach is the CO2 credits to be generated under the Kyoto Protocol(Wang et al., 2008). For example, CO2 can be instantly absorbed through bubbling it in a hy-droxide solution at 40�Celsius, producing sodium or ammonium bicarbonate. However, thedemand for these salts (although high—equivalent to around 14Gt/year) is supplied by theSolvay process, whereas a CO2 processwould require previous synthesis of sodiumhydroxide.

Biological CO2 mitigation has attracted a good deal attention as a strategic alternative.Microalgae cultivation gained importance because it associates CO2 mitigation and produc-tion of a wide range of commercial bioproducts.

Despite the fact that the existence of microalgae has been known for a long time, studies forits use as industrial microorganisms are relatively recent. Initial studies of microalgae culti-vation began in the late 1940s and early 1950s for its potential as a source of food. Concernsabout water pollution in the 1960s increased interest in the use of microalgae in wastewatertreatment. The perception in the 1970s that fossil fuels would run outmade thesemicroorgan-isms a focus of renewable fuel production. In the 1980s microalgae were used as a source ofvalue-added products, specifically nutriceuticals. In the late 1980s the low cost of oil causeda loss of interest in microalgae-based energy, whereas research with nutraceuticals andbiomass for feed continued. In the 2000s, global warming concerns associated with highoil prices made microalgal bioenergy projects popular again.

To create microalgal products, it is necessary to develop mass-cultivation techniques andto understand the physiological characteristics of each strain. There have been extensive stud-ies on process optimization (media and physicochemical parameter optimization, screeningand isolation of high CO2 tolerants, search for new valuable products, optimization anddevelopment of new vessels and systems for cultivation, for example) to try to overcomethe economic issues faced in industrial-scale production of microalgae. Two other aspectsare gaining importance: the use of industrial residues (to reduce media costs) and the carbonmarket (carbon credits as an additional element in the economic evaluation of the process).

The evaluation of nutrient needs in microalgal cultures is an important tool in processdevelopment using residues (domestic or industrial), and the quantification of carbon dioxidefixation is of great industrial interest since carbon credits can be traded on the internationalmarket and companies may use the process as a marketing strategy.

The rate of carbon uptake is limited by themetabolic activity ofmicroalgae, which is in turnlimited by photosynthesis. The ability to identify rates of consumption of nutrients is thus ofconsiderable importance to the understanding of the metabolism of microalgae and to avoidproblems in industrial cultivation of such microorganisms.

4.1.1 Microalgal Metabolism

Microalgae are a very heterogeneous group of microorganisms. The term microalgaeincludes prokaryotes and eukaryotes. Cyanobacteria (blue-green algae) are frequently unicel-lular, with some species forming filaments or aggregates. The internal organization of a

694.1 INTRODUCTION

cyanobacterial cell is prokaryotic, where a central region (nucleoplasm) is rich in DNA anda peripheral region (chromoplast) contains photosynthetic membranes. The sheets of thephotosynthetic membranes are usually arranged in parallel, close to the cell surface. Eukary-otic autotrophic microorganisms are usually divided according to their light-harvesting pho-tosynthetic pigments: Rhodophyta (red algae), Chrysophyceae (golden algae), Phaeophyceae(brown algae), and Chlorophyta (green algae). Their photosynthetic apparatus are organizedin special organelles, the chloroplasts, which contain alternating layers of lipoprotein mem-branes (thylakoids) and aqueous phases (Staehelin, 1986).

All photosynthetic organisms contain organic pigments for harvesting light energy. Thereare three major classes of pigments: chlorophylls (Chl), carotenoids, and phycobilins. Thechlorophylls (green pigments) and carotenoids (yellow or orange pigments) are lipophilicand associated in ChI-protein complexes, while phycobilins are hydrophilic. Chlorophyllmolecules consist of a tetrapyrrole ring (polar head, chromophore) containing a centralmagnesium atom and a long-chain terpenoid alcohol. Structurally, the various types ofChl molecules, designated a, b, c, and d, differ in their side-group substituent on the tetrapyr-role ring. All ChI have two major absorption bands: blue or blue-green (450–475 nm) and red(630–675 nm) (Niklas Engstrom, 2012). Chl a is present in all oxygenic photoautotrophs.

Photoautotrophic cultures seldom reach very high cell densities; they are more than anorder of magnitude less productive than many heterotrophic microbial cultures, the reasonthatmicroalgal cultures are carried in very large volumes.However, themicroalgal photosyn-thetic mechanism is simpler than that of higher plants, providing more efficient solar energyconversion. This makes microalgae the most important carbon-fixative group and oxygenproducer on the planet. Microalgae cultures have some advantages over vascular plants(Benemann andOswald, 1996): All physiological functions are carried out in a single cell, theydo not differentiate into specialized cells, and they multiply much faster.

4.1.2 Photosynthesis

Photosynthesis can be defined as a redox reaction driven by light energy, in which carbondioxide and water are converted into metabolits and oxygen. Photosynthesis is traditionallydivided into two stages, the so-called light reactions and the dark reactions. The first process isthe light-dependent process (light reaction), which occurs in the grana and requires the directenergy of light to make energy carrier molecules that are used in the second process. Thelight-independent process (or dark reaction) occurs in the stroma of the chloroplasts, wherethe products accumulated in the products of the light reaction are used to form C-C covalentbonds of carbohydrates. The dark reactions can usually occur if the energy carriers from thelight process are present.

In the light reactions, light strikes chlorophyll a in suchawayas to excite electrons to ahigherenergy state. In a series of reactions, the energy is converted (along an electron transportprocess) into ATP andNADPH.Water is split in the process, releasing oxygen as a byproductof the reaction. The ATP and NADPH are used to make C-C bonds in the dark reactions.

In the dark reactions, carbon dioxide from the atmosphere (or water for aquatic andmarineorganisms) is captured and reduced by the addition of hydrogen to form carbohydrates([CH2O]n). The incorporation of carbon dioxide into organic compounds is known as carbonfixation. The energy comes from the first phase of the photosynthetic process. Living systems


cannot directly utilize light energy, but they can, through a complicated series of reactions,convert it into C-C bond energy that can be released by glycolysis and other metabolicprocesses. So, the main role of the light reactions is to provide the biochemical reducing agentNADPH2 and the chemical energy carrier (ATP) for the assimilation of inorganic carbon, aspresented in Equation 4.1:

2NADPþ 3H2Oþ 2ADPþ 2Pi $ 2NADPH2 þ 3ATPþO2 ð4:1ÞThe fixation of carbon dioxide happens in the dark (in the stroma of chloroplasts) using the

NADPH2 and ATP produced in the light reaction of photosynthesis (Equation 4.2):

CO2 þ 4Hþ 2NADPHþ 3ATP $ CH2Oð Þ ð4:2ÞCarbon dioxide is available in water in three different forms: CO2, bicarbonate (HCO�

3 ), or
carbonate (HCO3
2�) (Figure 4.1), the relative amounts of which are pH dependent. Althoughplants and algae are known to be dependent exclusively on the Calvin-Benson-Bassham cycle(also known as the Calvin cycle) (Atomi, 2002), six autotrophic carbon-fixation pathways areknown. These are (1) the Calvin cycle, (2) the acetyl-CoA pathway, (3) the 3-hydroxypropionatecycle, (4) thereverse tricarboxylicacidcycle, (5)3-Hydroxypropionate/4-hydroxybutyratecycle,and (6)Dicarboxylate/4-hydroxybutyrate cycle (GeorgeFuchs, 2011). This sectiondiscusses theCalvin cycle, which is the most important in microalgae.

In the Calvin cycle there is only one enzyme responsible for CO2 fixation: ribulose1,5-biphosphate carboxylase/oxygenase, also known as Rubisco. Figure 4.2 shows the Calvin

CO2(g) CO2(aq) H2CO3 HCO3- CO3

2- FIGURE 4.1 Different forms inwhich carbon dioxide isavailable in water.

3 Ribulose-bis-P

3ADP + 3P

3ATP

6 Glycerate-P3 Ribulose-P

5 Glyceraldehyde-P

6 Glyceraldehyde-P

Organic compounds production

6ATP

6ADP + 6P

6NADPH

6NADP+ + 6P

6 Glycerate bis-P

CALVIN CYCLE

CO2FIGURE 4.2 The dark process ofCO2 capture and transformationthrough metabolism of photosyn-tethic microalgae (modified from

Masojıdek et al., 2004).

714.1 INTRODUCTION

cycle, where one molecule of ribulose 1,5-biphosphate and a CO2 are converted into twoglycerate phosphate. CO2 diffuses through the cell and is captured by the enzyme ribulosebiphosphate (Rubisco).

CO2 g� � $ CO2 aqð Þ $ H2CO3 $ HCO�

3 $ CO32�

The fixation of CO2 occurs in four distinct phases (Masojıdek et al., 2004):

1. Carboxylation. A reaction whereby CO2 is added to the five carbon sugar ribulosebisphosphate (Ribulose-bis-P) to form two molecules of phosphoglycerate (Glycerate-P).This reaction is catalyzed by the enzyme ribulose biphosphate carboxylase/oxygenase(Rubisco).

2. Reduction. To convert Glycerate-P into 3-carbon sugars (Triose-P), energymust be added inthe form of ATP and NADPH2 in two steps, which are the phosphorylation of Glycerate-Pto form diphosphoglycerate (Glycerate-bis-P) and the reduction of Glycerate-bis-P tophosphoglyceraldehyde (Glyceraldehyde-P) by NADPH2.

3. Regeneration. Ribulose-P is regenerated for further CO2 fixation in a complex series ofreactions combining 3-, 4-, 5-, 6-, and 7-carbon sugar phosphates, which are not explicitlyshown in the diagram.

4. Production. The primary end products of photosynthesis are considered to be carbohydrates,fatty acids, amino acids, and organic acids.

Besides the carboxylase activity described here, all Rubiscos (there is more than one type)are known to display an additional oxygenase activity in which an oxygen molecule, com-peting with CO2 for the enzyme-bound eno-diolate of RuBP, reacts with RuBP to form3-phosphoglycerate and phosphoglycolate (Atomi, 2002). The latter product is subse-quently oxidatively metabolized via photorespiration, leading to a net loss in carbon dioxidefixation. Photorespiration thus represents a competing process to carbon fixation, where theorganic carbon is converted into CO2 without any metabolic gain. Photorespiration dependson the relative concentrations of oxygen and CO2 where a high O2/CO2 ratio stimulates thisprocess, whereas a low O2/CO2 ratio favors carboxylation. Rubisco has low affinity by CO2;its Km (half saturation) is approximately equal to the level of CO2 in air. Thus, under highirradiance, high oxygen level, and reduced CO2, the reaction equilibrium is shifted towardphotorespiration. For optimal yields in microalgal mass cultures, it is necessary to minimizethe effects of photorespiration, achieved by an effective stripping of oxygen and by CO2

enrichment. For this reason, microalgal mass cultures are typically grown at a much higherCO2/O2 ratio than that found in air, which is in turn an opportunity to reuse industrialgas emissions.

The source of nitrogen in cultivation of microalgae seems to cause changes in oxygenproduction during photosynthesis. The ratio between O2 evolution rate and CO2 uptake rate(the photosynthetic quotient, PQ) depends on the composition of the produced biomass andthe substrates that are used. Especially oxidized nitrogen sources, which must be reducedbefore they are incorporated into the biomass, affect the PQ. When nitrate is used, it isexpected at an evolution of 1.3 mol O2 per mol of CO2 assimilated, whereas nitrite promotesa release of 1.2 mol O2 and ammonia 1.0 mol O2 (Eriksen et al., 2007). Approximately 20%of O2 evolution equivalents can be accounted for by NO3

� uptake and assimilation underN-replete conditions (Turpin, 1991).


4.1.3 Microalgae Culture Fundamentals

Studies on microalgae are preferably done under controlled conditions. Microalgae biore-actors are often designed differently from bioreactors used to grow other microorganisms.Two parameters are the most important in algae cultivation: efficiency of light utilizationand availability of dissolved CO2.

Like any organism, microalgae have nutritional requirements: carbon sources, energy,water, and inorganic nutrients. In the case of microalgae, the carbon source can be CO2

and the energy comes from sunlight. As microalgae grow in aqueous suspension, the manip-ulation and control of culture conditions makes their cultivation feasible, thus the productiv-ity is limited mostly by the available of light. Responses by algal cells to nutrients andcultivation environments can be used to manipulate the processes to favor the productionof algal biomass (Benemann et al., 2002).

The development of media for microalgae cultivation involves a sufficient carbon source(carbon is a part of all the organic molecules in the cell, making up as much as 50% of thealgal biomass); salt concentration (depending on the original biotope of the alga); nitrogen(represents about 5–10% of microalgae dry weight); phosphorus (part of DNA, RNA, ATP,cell membrane); sulfur (constituent of amino acids, vitamins, sulfolipids and is involved inprotein biosynthesis); potassium (cofactor for several enzymes and involved in protein syn-thesis and osmotic regulation); magnesium (the central atom of the chlorophyll molecule);iron (constituent of cytrochromes and important in nitrogen assimilation); pH of themedium; temperature; trace elements, and addition of organic compounds and growthpromoters.

Carbon is important because it is the source of energy for many cellular events (such asmetabolites production) and reproduction and is part of the physical structure of the cell.In conditions of low dissolved inorganic carbon (DIC), a DIC transport is induced in mostmicroalgae (Matsuda and Colman, 1995), allowing normal cell growth.

Depending on thematerial used in cultivation ofmicroalgae and the utilization of biomass,three different systems can be distinguished (Becker, 1994):

1. Systems in which a selected algal strain is grown in a so-called clean process, using freshwater, mineral nutrients, and carbon sources. The algae in such systems are intended to beutilized mainly as food supplements.

2. Systems using sewage or industrial wastewater as the culture medium. The cultivation ofthe microalgae involves secondary (BOD removal) and tertiary (nutrient removal)treatments and production of biomass-based products.

3. Cultivation of algae in enclosed systems under sunlight or artificial light, with cellspreferably being grown in autotrophic media.

Microalgae are microorganisms that are capable of producing many different compoundsof industrial interest, some with high and some with low aggregated value. The final value ofthe product and its destination directly influence the conditions of cultivation. Therapeuticalcompounds produced by microalgae, for example, must be produced through a totally con-trolled and clean process, whereas for the fuel industry residues can be used and the controlof the process can be less accurate. The low culture concentration and the corresponding highdownstream costs define production trends.

734.2 CARBON DIOXIDE FIXATION BY MICROALGAE

The utilization of complex media (those of which the composition is not determined, suchas industrial residues) in the cultivation of microalgae is one alternative to make the produc-tion of some microalgal metabolites economically feasible. Associated with residue compo-sition and microalgae metabolism, knowledge of the needs of the microalgae might savetime (and money) in the development of a process. It is very important to supply allmicroalgae chemical needs because it is known that variations in the chemical compositionof phytoplankton are also tightly coupled to changes in growth rate (Goldman et al., 1979).


4.2.1 Carbon Dioxide’s Role in Photobioreactors

An important issue inmost photobioreactors and the first step inCO2 fixation is the diffusionof CO2 from the gas phase to the aqueous phase. The solubility of CO2 in the culture media de-pends on depth of the pond, the mixing velocity, the productivity of the system, the alkalinity,and theoutgassing. It has been reported (Becker, 1994) thatonly 13–20%of the suppliedCO2wasabsorbed in racewaypondswhenCO2 gaswas bubbled into the culture fluid as a carbon source.Binaghietal. (2003)achievedamaximumvalueof38%efficiencyofcarbonutilization inSpirulinacultivation.Gas–liquidcontact timeandgas–liquid interfacial areaare, therefore, twokey factorsto enhance the gas–liquidmass transfer. In addition, high oxygen tension is problematic, since itpromotes CO2 outgassing and competes with CO2 for the CO2-fixing enzyme (RuBisCO).

The capacity for carbon dioxide storage in a growth medium is important because it deter-mines the amount of CO2 thatmay be used formedium saturation, leading to high growth ratesand in-process economics. Since CO2 reacts withwater, producing carbonic acid and its anions,chemical equilibriumwill have a significant impact on the amount of carbondioxide stored. pHis themajor determinant of the relative concentrations of the carbonaceous system inwater andaffects the availability of carbon for algal photosynthesis in intensive cultures (Azov, 1982).

The absorption of CO2 into alkaline waters may be accelerated by one of two majoruncatalyzed reaction paths: the hydration of CO2 and subsequent acid-base reaction to formbicarbonate ion, and the direct reaction of CO2with the hydroxyl ion to form bicarbonate. Therate of the former reaction is faster at pH values below 8, whereas the latter dominates abovepH 10. Between pH 8 and 10, both are important.

Microalgae can fixate carbon dioxide from different sources, including CO2 from the atmo-sphere, from industrial exhaust gases (e.g. furnaces flue gases), and in form of soluble carbon-ates. Traditionally, microalgae are cultivated in open or closed reactors and aeratedwith air orair enriched with CO2. Industrial exhaust gases contain up to 15% of carbon dioxide in theircomposition, being a rich (and cheap) source of carbon for microalgae growth.

In microalgae cultivation, high concentrations of CO2 are not usually used because it mayresult in decreasing the pH, since unutilized CO2 will be converted to HCO�

3 . Shiraiwa et al.(1991) and Aizawa and Miyachi (1986) reported that an increase in CO2 concentration of sev-eral percent resulted in the loss of a carbon concentration mechanism (CCM), and any furtherincrease was always disadvantageous to cell growth. Most processes use air enriched withCO2 (2–5% CO2 final concentration), but some studies using high CO2-resistant strains arebeing described in scientific literature.


If there is not enough CO2 gas supply, algae will utilize (bi)carbonate to maintain itsgrowth. When algae use CO2 from bicarbonate, an increase of pH is observed (a growth in-dicator), even reaching growth-inhibition pH values. To overcome pH fluctuation, the CO2

gas injection should be controlled in such a way that photosynthesis rates are balanced withenough and continuous availability of dissolved carbon. Interesting studies about isolationand selection of strains with high CO2 absorption capacity, which is an important step nomatter the process in development, are available in scientific literature. Maintaining constantCO2-free concentration in the media will keep carbon uptake constant.

The ability to accumulate DIC has been shown to occur in many algae and cyanobacteria(Williams and Colman, 1995). Whereas CO2 can diffuse into algal cells and is the substrate forcarbon fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubiscO), it forms asmall proportion of the total available inorganic carbon. The largest proportion of totalDIC available to microalgae consists of ionic HCO�

3 , which has a low capacity for diffusionacross cell membranes (Young et al., 2001). A number of eukaryotic microalgae have devel-oped mechanisms that permit the use of HCO�

3 for photosynthesis (Miller and Canvin, 1985).Access to the larger pool of HCO�

3 is assumed to involve one or both of two basic processes:

1. In some green algae, the use of HCO�3 has been correlated with the presence of external

carbonic anhydrase (CA) activity (Aizawa and Miyachi, 1986). In these cases externalCA is thought to facilitate the use of HCO�

3 by maintaining equilibrium between HCO�3

and CO2, and thereby maintaining the supply of CO2 to a specific transporter (Aizawaand Miyachi, 1986).

2. Direct HCO�3 transport via a transmembrane bicarbonate transporter, which has been

demonstrated even in cells that have external CA activity (Williams and Turpin, 1987).The involvement of transmembrane ATPase proteins was also reported in DIC uptake bychlorophytes (Ramazanov et al., 1995).

4.2.2 Methods of CO2 Fixation Quantification

Since outdoor sunlight cannot be controlled, carbon fixation by microalgae is usuallystudied indoors under artificial illumination. A good deal of scientific effort is being madeto evaluate microalgae CO2 fixation potential. Most of these efforts focus the fixation intobiomass (Chae et al., 2006; Jacob-Lopes et al., 2008; Kajiwara et al., 1997). However, thesestudies did not quantify the total carbon dioxide fixed effectively by microalgae (Jacob-Lopeset al., 2008; Fan et al., 2007), since there are other routes for carbon besides biomass generation,such as mineralization (formation of soluble bicarbonate and carbonate) and production ofextracellular products such as polysaccharides, volatile organic compounds (Shaw et al.,2003), organohalogens (Scarratt and Moore, 1996), hormones, and others.

The determination of global rates of carbon dioxide sequestration throughmass balances ofCO2 in the liquid or gas phase of the systems (Eriksen et al., 2007) gives more complete data.One approximation for the rates may be obtained by evaluating dissolved inorganic carbonconcentration in the culture media while monitoring the pH variation (see methodology atValdes et al., 2012). This shows that carbon fixation by microalgae is a complex processwhereby biomass productionmight be a part of the total carbon destination. In addition, littleinformation is available with respect to the simultaneous research of both the global rates of

5

4.5

4

3.5

3

2.5

2

1.5

CO

2 co

ns

1

0.5

01 25 49 73 97 121 145 169

Time (hours)

193 217 241 265 289 313 337 361

O2

con

s

-3.5

-2.5

-1.5

-0.5

0.5

0

1

1.5

2

-3

-2

-1

CO2 consumed (g/h) CO2 base line (g/h)

O2 Base Line (g/h)O2 consumed (g/h)

FIGURE 4.3 Gas phase analysis carried by Sydney et al. (2011) showing the carbon consumption and oxygenproduction profiles.


carbon dioxide sequestration and the rates of incorporation of carbon into the microalgaebiomass (Chiu et al., 2008).

Sydney et al. (2011) studied the global CO2 fixation rate of four microalgae through a massbalance of the gas phase. The experiments were carried out in a photobioreactor coupledwithsensors tomeasure CO2 in the inlet and outlet gases. The net carbon dioxidemitigation duringeach microalgal cultivation was evaluated. Nutrient consumption, biomass production (andcomposition), and possible extracellular products were analyzed throughout the process.It was found that between 70% and 88% of the carbon dioxide consumedwas used in biomassproduction. This finding indicates that, to explore the whole potential of microalgal mitiga-tion capacity (considering negotiations in the carbonmarket), carbon balancemight be carriedthrough (complex) carbon balance in the gas phase. The problem is that it is difficult to carryout this kind of analysis in open photobioreactors and to standardize this methodology.Figure 4.3 presents the profile of carbon dioxide consumption obtained during gas phaseanalysis during cultivation. It is interesting to note that CO2 consumption (in blue) has a com-plementary behavior with O2 production due to photosynthesis and respiration processesduring light and dark cycles.

4.2.3 Carbon Fixation of Industrially Important Microalgae

Carbon fixation by microalgae is in vogue. In the last decade, more than 4,000 papers werepublished globally on this subject. Table 4.1 presents some rates of carbon dioxide describedin the literature.

Among all species of microalgae, four are most common industrially: Spirulina, Chlorella,Dunaliella, andHaematococcus. Despite not being used industrially, Botryococcus is also largelystudied due to its potential use as a source of hydrocarbons. These microalgae’s potential forcarbon fixation is discussed next.

TABLE 4.1 Data of Biomass Productivity and CO2 Fixation Rate from Microalgae.

Microalgae Strain

Biomass

(mg L�1 d�1)

CO2 Fixation

Rate

(mg L�1 d�1) Reference

Spirulina platensis 145 318 Sydney et al., 2011

Chlorella vulgaris 129 251 Sydney et al., 2011

Synechocystis aquatilis 30 50 Zhang et al., 2001

Anabena sp. 310 1450 Lopez et al., 2009

Botryococcus braunii 207 500 Sydney et al., 2011

Dunaliella tertiolecta 143 272 Sydney et al., 2011

Chlorococcum littorale 530 900 Kurano et al., 1996

Aphanothece microscopica Nageli 301 562 Jacob-lopes et al., 2009

Chlorella, Oscillatoria, Oedogonium, Anabaena,Microspora and Lyngbya (mixed culture)

131 161 Tsai et al., 2012


4.2.3.1 Chlorella vulgaris

The first photosynthetic microbe to be isolated and grown in pure culture was the fresh-water microalga Chlorella vulgaris. It is a spherical unicellular eukaryotic green algaethat presents a thick cell wall (100–200 nm) as its main characteristic. This cell wallprovides mechanical and chemical protection, and its relation to heavy metals resistance isreported, which explains why C. vulgaris is one of the most used microorganisms for wastetreatment.

The uptake of carbon by C. vulgaris cells is done through the enzyme carbonic anhydrase,which catalyzes the hydration of CO2 to form HCO�

3 and a proton. Hirata and collaborators(1996) studied carbon dioxide fixation by this microalga, which showed important variationscomparing cultivation under fluorescent lamps and sunlight. In the first case the estimatedrate of carbon dioxide fixation was 865 mg CO2 L

�1 d�1; in a sunlight regimen the estimatedrate achieved 31.8 mg CO2 L

�1 d�1. Winajarko et al. (2008) achieved a transferred rate of441.6 g CO2 L

�1 d�1 under the same cultivation conditions as Hirata et al. (1996). Accordingto Sydney et al. (2011), in experiments using classic synthetic media and a 12-h light/darkregimen, C. vulgaris biofixation rate of carbon dioxide is near 250 mg L�1 day�1.

Carbon fixation by Chlorella vulgaris is variable and depends, among other factors, on theconcentration of CO2 in the gaseous source. Yun et al (1997) cultivated C. vulgaris in 15%of carbon dioxide and achieved a fixation of 624 mg L�1 day�1; Scragg et al. (2002) achieveda fixation of 75 mg L�1 day�1 under CO2 concentration of 0.03%. In the same study, Scraggtested a medium with low nitrogen and the fixation rate was 45 mg L�1 day�1, suggestingthat nitrogen also influences carbon uptake rate.

Some studies (Chinassamy et al., 2009; Morais and Costa, 2007) indicate that the bestconcentration of CO2 in the gas supplied to C. vulgaris growth is about 6%.


4.2.3.2 Botryococcus braunii

Botryococcus is a colonialmicroalga that iswidespread in fresh and brackishwaters of all con-tinents. It is characterized by its slow growth and by containing up to 50% by weight of hydro-carbons.B. braunii is classified intoA, B, andL races,mainly basedon the difference between thehydrocarbonsproduced(MetzgerandLargeau,2005).Banerjeeetal. (2002)differentiate theracesas follows: Race A produces C25 to C31 odd-numbered n-alkadienes and alkatrienes; B race pro-ducespolymethylatedunsaturatedtriterpenes,calledbotryococcenes (CnH2n–10,n¼30–37); andLrace produces a single tetraterpene hydrocarbon C40H78 known as lycopadiene.

The cells of B. braunii are embedded in a communal extracellular matrix (or “cup”), whichis impregnated with oils and cellular exudates (Banerjee et al., 2002). B. braunii is capable ofsynthesizing exopolyssaccharides, as reported by Casadevall et al. in 1985. Higher growthand production of EPS, which ranges from 250 g m–3 for A and B races to 1 kg m–3 for theL race, occur when nitrate is the nitrogen source instead of urea or ammonium salts(Banerjee et al., 2002). Phosphorus and nitrogen are also important factors in accumulationof hydrocarbons by the microorganism (Jun et al., 2003).

The metabolic energy devoted to produce such large amounts of hydrocarbons makes thisspecies noncompetitive in open mass cultures, since strains not so burdened can grow muchfaster and soon dominate an outdoor pond culture (Benemann et al., 2002). B. braunii has beenreported to convert 3% of the solar energy to hydrocarbons (Gudin and Chaumont, 1984).Being synthesized by a photosynthetic organism, hydrocarbons from algae can be burnedwithout contributing to the accumulation of CO2 in the atmosphere.

Dayananda et al. (2007) cultivated Botryococcus braunii strain SAG 30.81 in shake flasks andobtained a maximum cell concentration of 0.65 g L�1 under 16:8 light:dark cycle. Experimentswith different strains of B. Braunii indicate that the biomass yield is inversely proportional tolipid accumulation. The maximum biomass yield achieved was 2 g L�1 (with 40% of lipids) andthe lowerwas 0.2 g L�1 (with 60%of lipids). Outdoor experimentswith thismicroalga achieved ahigh biomass yield of 1.8 g L�1 but a very low lipid accumulation. It was also showed byDayananda and collaborators that exopolyssaccharides production by Botryococcus braunii SAG30.81 is not affected by light regimen in MBM media, different from lipids and proteins pro-duction. Sydney et al. (2011) carried experiments with this same strain under 12 h light:dark cycle in 5%CO2enriched air andachievedahighbiomassproductionof 3.11 g L�1with 33%lipids in 15 days. Carbon dioxide fixation rate was calculated as near 500mg L�1 day�1. B. brauniibiomasscompositionalso included39%proteins,2.4%carbohydrates,13%pigments,and7.5%ash.

Marukami and Ikenouochi (1997) achieved a carbon dioxide fixation greater than 1 gramper liter by Botryococcus braunii cultivated for hydrocarbon accumulation.

4.2.3.3 Spirulina platensis

Spirulina are multicellular ilamentous cyanobacteria actually belonging to two separategenera: Spirulina and Arthrospira. These encompass about 15 species (Habib et al., 2008). Thismicroorganism grows in water, reproduces by binary fission, and can be harvested andprocessed easily, having significantly high macro- and micronutrient contents. Their mainphotosynthetic pigments are chlorophyll and phycocyanin. The helical shape of the filaments(or trichomes) is characteristic of the genus and is maintained only in a liquid environment orculture medium.


Spirulina is found in soil, marshes, freshwater, brackish water, seawater, and thermalsprings. Alkaline, saline water (>30 g/L) with high pH (8.5–11.0) favors good productionof Spirulina, especially where there is a high level of solar radiation. It predominates in higherpH and water conductivity. Like most cyanobacteria, Spirulina is an obligate photoautotroph,i.e., it cannot grow in the dark on media containing only organic carbon compounds.It reduces carbon dioxide in the light and assimilates mainly nitrates.

Spirulina contains unusually high amounts of protein, between 55% and 70% by dryweight, depending on the source. It has a high amount of polyunsaturated fatty acids(PUFAs), 30% of its 5–6% total lipids, and is a good source of vitamins (B1, B2, B3, B6, B9,B12, C, D, E). Spirulina is a rich source of potassium and also contains calcium, chromium,copper, iron, magnesium, manganese, phosphorus, selenium, sodium, and zinc. Thesebacteria also contain chlorophyll a and carotenoids.

The optimum pH of the Spirulina sp. culture is between 8.5 and 9.5 (Watanabe et al., 1995).Cyanobacteria possess a CO2-concentating mechanism that involves active CO2 uptake andHCO�

3 transport. In experiments conducted by Morais and Costa (2007), carbon fixation interms of biomass by Spirulina platensiswas estimated in 413 mg L�1 d�1, near those achievedby Sydney et al. (2011).

4.2.3.4 Dunaliella sp.

Dunaliella isabiflagellateunicellulargreenalga.Cellsareround-shapedandfoundinbrackishenvironments; it is a motile species and has a high tolerance for salt, temperature, and light.Motion of cells is important since it facilitates nutrient transport, especially in poor-nutrientwaters.Dunaliella species are relatively easy to culture. The cell divides by simple binary fission,and no evidence of cell lysis, encystment, or spore formation is observed (Segovia et al., 2003).

Dunaliella thrives over a wide pH range and expresses a capacity for extremely efficient DICaccumulation, incorporating a capacity to use HCO�

3 in addition to CO2 (Aizawa andMiyachi,1986; Young et al., 2001). Kishimoto et al. (1994) cultivated a Dunaliella strain for pigment pro-ductionwith 3% of CO2 and achieved a carbon uptake of 313mg L�1 day�1. Sydney et al. (2011)cultivated a D. tertiolecta strain and achieved a CO2 fixation rate of 272 mg L�1 day�1.

Dunaliella is an important microalgae for industrial processes since it produces a widevariety of commercial products (mainly pigments) and the rupture of the cells is very easy.b-carotene large-scale production facilities are in operation around theworld (Hawaii, UnitedStates, Australia, Japan).

4.2.3.5 Haematococcus sp.

Haematococcus is a green algae (Chlorophyta), mobile, single-celled, and capable of synthe-sizing and accumulating the pigment astaxanthin in response to environmental conditions,reaching from 1.5% up to 6% by weight astaxanthin (Vanessa Ghiggi, 2007). The astaxanthinproduced by Haematococcus pluvialis is about 70% monoester, 25% diesters, and 5% free(Lorenz and Cysewski, 2000).

These algae, however, have some undesirable characteristics compared to othermicroalgae grown successfully on a commercial scale. The biggest concern is mainly relatedto a relatively slow growth rate, allowing easy contamination. Therefore, many studies havesought to improve the low rate of growth of vegetative cells, which is, exceptionally,1.20 div/day (Gonzales et al., 2009).

794.3 PRACTICAL ASPECTS OF MASS CULTIVATION FOR CO2 FIXATION

Alternatively, its mixotrophic (Guerin et al., 2003; Gonzales et al., 2009) and heterotrophic(Hata et al., 2001) metabolism, using acetate as carbon source, has also been studied anddocumented; however, these conditions have not been applied to commercial-scale culturesand are not interesting in terms of carbon fixation.

4.3 PRACTICAL ASPECTS OF MASS CULTIVATION FORCO2 FIXATION

4.3.1 Cultivation Vessels

Many different configurations of photobioreactors are possible: from simple unmixedopen ponds to highly complex enclosed ones. The configuration of the bioreactor has greatinfluence on carbon dioxide consumption during algal growth. Most of the recent researchin microalgal culturing has been carried out in photobioreactors with external light supplies,large surface areas, short internal light paths, and small dark zones. Examples include openponds (the cheapest ones), tubular reactors, flat panel reactors, and column reactors (stirred-tank reactors, bubble columns, airlift).

The applications of such systems range from the small-scale production of high-valueprod-ucts to the large-scale production of biomass for feed. The choice between the different designsof photobioreactors must be specific to the intended application and local circumstances.

Open ponds can be an important and cost-effective component of large-scale cultivationtechnology, and optimal design parameters have been known for many years. The elongated“raceway type” of open pond, using paddlewheels for recirculation and mixing, was devel-oped in the 1950s by the Kohlenbiologische Forschungsstation in Dortmund, Germany.However, sustained open pond production proved to be feasible for only three microalgae:Spirulina platensis, Dunaliella salina, and fast-growing Chlorella, in all cases because con-tamination by other species can be avoided.

Beyond the economical difference between the types of photobioreactors feasible for algaecultivation, light incidence and CO2 availability are the two main factors influencing algaegrowth. Large surface areas are essential to ensure enough light diffusion to the media,but they are normally associated with very little time to mass transfer the gas to the liquidphase (short liquid column). The optimal condition of light diffusion and CO2 availabilityis easily achieved in a closed reactor for logical reasons: In open photobioreactors, theundissolved CO2 is lost to the atmosphere, whereas in closed ones it is possible to increase(and maintain) partial pressure.

4.3.2 Light Diffusion

Themost important parameter considered for the development and utilization of a specifictype of reactor for microalgae cultivation is the light diffusion. The productivity of photo-autotrophic cultures is primarily limited by the supply of light and suffers from lowenergy-conversion efficiencies caused by inhomogeneous distribution of light inside thecultures (Grobbelaar, 2000). At culture surfaces, light intensities are high, but absorptionand scattering result in decreasing light intensities and complex photosynthetic productivityprofiles inside the cultures (Ogbonna and Tanaka, 2000). High light intensities at culture


surfaces may cause photoinhibition, and the efficiency of light energy conversion intobiomass (photosynthetic efficiency) is low. An overdose of excitation energy can lead to pro-duction of toxic species (e.g., singlet oxygen) and to photosynthesis damage (Janssen, 2002)

Byminimizing depth, volume is reduced or area is increased, light diffusion is maximized,and so is cell concentration. From common types of photobioreactors, light paths in openponds are usually 10–30 cm depth, in tubular reactors ranges from 1–5 cm, and in flat panelreactors from 2–5 cm.

The light regimen itself is influencedbyincident light intensity, reactordesignanddimension,celldensity,pigmentationof thecells,mixingpattern,andmore. Inoutdoorphotobioreactors thelight regimen is also influenced by geographical location, time of day, and weather conditions.Nowadays, open paddlewheel-mixed pond is the most commonly used photobioreactor.

Some studies discuss the effect of mixing and productivity due to the “flashing-light”effect: A few milliseconds’ flashes of high light intensity followed by a several-fold longerperiod of darkness do not reduce culture productivity from those under constant illumination(Kok, 1953). This effect is not observed in ponds, where the light/dark period is longer. Forexample, although light/dark cycles of 94/94 ms were sufficiently short to increase the pho-tosynthesis efficiency in cultures of Dunaliella tertiolecta, light/dark cycles of 3/3 s were toolong and the PE decreased in comparison to continuously illuminated cultures (Janssen et al.,2001). This refers to the theory of photosynthesis, inwhich carbon fixation is not dependent onthe presence of light because sufficient energy has been absorbed.

4.3.3 Mixing

To optimize the photosynthesis rate and gas solubility in the media, mixing is very impor-tant. Besides that, mixing is important for homogeneous distribution of cells, metabolites, andheat and to transfer gases across gas–liquid interfaces. Mixing can be done mechanically bypaddlewheel in raceways (Figure 4.4) or by gas flow in bubble columns.

FIGURE 4.4 Paddlewheel mixing of racewayponds at Ouro Fino Agronegocio (Brazil).

814.4 CARBON MARKET FOR MICROALGAL TECHNOLOGIES


The Kyoto Protocol invented the concept of carbon emissions trading in a flexible mech-anism whereby developed countries could use carbon credits to meet their emission reduc-tion commitments. The world carbon market is based on a cap-and-trade system. Accordingto Mark Lazarowicz (2009), under cap-and-trade, a cap is set on emissions, as explained fur-ther by the author: “Allowances are provided, either through purchase or allocation, to emit-ters covered by the cap. These emitters are required to submit allowances equal to the amountof greenhouse gases emitted over a predetermined period. The difference between expectedemissions and the cap creates a price for the allowances. Emitters who can reduce emissionsfor less than the price of an allowance will do so. If, however, abatement costs more than theprice of an allowance, it makes sense to purchase the allowance. The transfer of allowances isthe ‘trade.’ The relative difficulty of abatement or scarcity of allowances sets the price ofcarbon. In theory, those that can reduce emissions most cheaply will do so, achieving thereduction at the lowest possible cost.” For this reason, the carbon market seems to be a tem-porary alternative while cleaner technologies are developed, including new ones andimprovement of the existing ones.

The carbon market jumped from $63 billion in 2007 to $126 billion in 2008, which meansalmost 12 times the value of 2005, according to the World Bank report of 2009. Credits weresold for 4.8 billion tons of carbon dioxide, a value 61% higher than that of the previous year.By 2020 the market could be worth up to $2–3 trillion per year (Point Carbon, Carbon MarketTransactions in 2020: Dominated by Financials?, May 2008).

Theworld carbonmarket ismainly dependent on energy-use policies. The focus is to replaceexisting high dependence on fossil fuels with renewable ones; around 90% of total global CO2

emissions are from fossil fuel combustion (excluding forest fires and woodfuel use; Olivieret al., 2011). The principal technical means of reducing fossil fuel consumption (and conse-quently emissions) are substituting fossil fuels with renewable or less carbon-content sourcesof energy and improving energy efficiency. Renewable energy’s share of the global energysupply increased from 7% in 2004 to over 8% by 2009 and 2010 (Olivier et al., 2011).

According to the “Long-term trend in global CO2 emission, 2011 report,” total global CO2

emissions had increased 30% since 2000, to 33 billion tones, and 45% since 1990, the base yearof the Kyoto Protocol. In 1990 the industrialized countries, with a mitigation target for totalgreenhouse gas emissions under the Kyoto Protocol (including the United States, which didnot ratify the protocol), had a share in global CO2 emissions of 68% versus 29% for developingcountries. In 2010 the large regional variation in emission growth trends resulted in shares for54% of developing countries and 43% for mature industrialized countries.

Microalgae can play a very interesting role in this context. While fixating carbon duringgrowth (to be traded in the market), some species can accumulate lipids, which can be usefor direct combustion or transformed in biodiesel to replace fossil sources. This is one ofthe developing technologies that receives more attention from the scientific communityaround the world.

The carbon market for microalgal carbon mitigation processes is a big challenge. Its en-trance in this market will coexist with other renewable energy technologies that are receivinglots of investment, which means that it must be more advantageous or differentiated. Tradesof carbon papers are carried mainly based on agriculture and forestry (reforestation, land


management, reduced emissions from deforestation). Great efforts are being made in the de-velopment and implantation of renewable energy technologies (wind power, solar photovol-taic, and vegetable-based biodiesel technologies).

In terms of development of more efficient and sustainable industrial processes, microalgaecan play an interesting role through combining the use of domestic and industrial wastewater(mainly that lacking fermentable carbon) and industrial gaseous wastes with cogeneration ofvaluable products, reducing carbon emissions and generating tradable carbon papers.According to the mass balance (Equation 4.3), where the biomass composition is given asCH1.78 N0.15O0.52 (analysis made in CHNS analyzer carried at the Bioprocess Engineeringand Biotechnology Department, Federal University of Parana, Brazil), around 1.8 gCO2 is con-sumed for each gram of dry biomass produced during microalgal growth. This means that,for producing 1 Carbon Paper (1 ton CO2), an area less than 1,000 square meters is needed(considering a biomass concentration in the culture of 3 g L�1 and a pond with 20 cm highof liquid).

0:815 H2Oþ CO2 þ 0:15 HNO3 ! CH1:78 N0:15O0:52 þ 1:37 O2 ð4:3Þ

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